il-1β,il-18,tnf-α,il-8 · 2020. 8. 8. · research in takanawa clinic and receive advisory fees...
TRANSCRIPT
medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20146175.this version posted July 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.
1
IL-1β,IL-18,TNF-α,IL-8は新型コロナウイルスが感染した際にあがるサイトカイン。清肺肺毒
湯は新型コロナウイルスに感染したような擬似状態を作り出す。そして実際に新型コロナに
感染した際に自然免疫が最大に機能する準備を整える予防的働きをする可能性が高い。
Qing Fei Pai Du Tang, a Chinese multi-herbal medicine formulated against COVID-19,
elevates the plasma levels of IL-1β, IL-18, TNF-α, and IL-8
Yasunari Kageyama1, 2, Koichi Aida1, Kimihiko Kawauchi1, Masafumi Morimoto1, Tomoka
Ebisui1, Tetsu Akiyama3, Tsutomu Nakamura1, 3
1Takanawa Clinic, 4-23-6, Takanawa, Minato-ku, Tokyo, 108-0074, Japan
2Tokai University Hospital, 143 Shimokasuya, Isehara-shi, Kanagawa, 259-1193, Japan
3Laboratory of Molecular and Genetic Information, Institute for Quantitative Biosciences,
The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan
Corresponding Author
Tsutomu Nakamura
Laboratory of Molecular and Genetic Information
Institute for Quantitative Biosciences, The University of Tokyo
1-1-1, Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan
Tel: +81-3-5841-7834
Fax: +81-3-5841-8482
E-mail: [email protected]
medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20146175.this version posted July 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.
2
Abstract
There are currently no specific vaccine or drugs proven to be effective against COVID-19.
Traditional Chinese herbal medicine has been integrated into the official therapeutic protocol
against COVID-19 in China. Qing Fei Pai Du Tang (QFPDT) is a Chinese multi-herbal
formula newly developed and specifically optimized for the treatment of COVID-19.
Therapeutic administration of QFPDT resulted in an improved cure rate in mild to
criticallyill patients. However, the immunological mechanism for the efficacy of QFPDT has
been poorly understood. Furthermore, the feasibility of prophylactic use in uninfected
individuals remain unconfirmed. We thus examined whether the administration of QFPDT at
a dose lower than recommended for therapeutic use alters hematological and/or
immunological measures in healthy individuals. We found that QFPDT elevates the plasma
levels of IL-1β, IL-18, TNF-α, and IL-8, which are key mediators of acute inflammatory
responses to ssRNA viruses. No apparent adverse effects were observed during the trial. Our
finding suggests that the pharmacological action of QFPDT is associated with the
upregulation of a subset of proinflammatory cytokines despite its clinical benefits for
COVID-19 patients. We should therefore be careful in its prophylactic use in uninfected
individuals until we have a better understanding of the immunopharmacological action of
QFPDT through further clinical studies with larger cohorts.
Keywords
COVID-19, IL-18, IL-1β, IL-8, Qing Fei Pai Du Tang, TNF-α
Introduction
Currently effective vaccines or specific therapeutic agents against COVID-19 remain
unavailable. The majority of patients with COVID-19 in China have been treated with a
medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20146175.this version posted July 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.
3
combination of traditional Chinese and modern Western medicine [1–3]. The use of several
Chinese herbal formulas is encouraged for the treatment of COVID-19 in the latest version
of the Diagnosis and Treatment Protocol for Novel Coronavirus Pneumonia (Trial Version 7)
released by the National Health Commission of China. One of them is “Qing Fei Pai Du
Tang” (QFPDT, the Chinese word for “lung cleansing and detoxifying decoction”), which is
newly formulated and specifically optimized for the symptoms of COVID-19. QFPDT has
shown a satisfactory therapeutic efficacy when administered to thousands of mild to critical
cases [1–3]. However, the underlying pharmacological mechanism and the feasibility of
prophylactic application to uninfected individuals remain unknown. We thus examined
whether QFPDT administration at a dose lower than recommended for therapeutic use
affects hematological and immunological measures in healthy individuals.
Materials and Methods
Statement of Ethics
This study was carried out in accordance with The Code of Ethics of the World Medical
Association (Declaration of Helsinki). All procedures were approved by the Ethics
Committees of the Takanawa Clinic (approval number: 2020-2). A signed informed consent
form was obtained from each participant prior to inclusion in this study. All experimental
procedures and data analyses were conducted by investigators who were blinded to the
subjects’ clinical information using a de-identified dataset. This study has been registered on
University Hospital Medical Information Network-Clinical Trials Registry (UMIN-CTR)
under the trial number UMIN000040341.
Subjects
This is an open-label, single-arm study to evaluate the feasibility of prophylactic use of
QFPDT in healthy individuals and obtain a clue to the pharmacological action of QFPDT. A
medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20146175.this version posted July 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.
4
total of 18 healthy volunteers were enrolled (5 males, 13 females; ages 22–58 years; mean
age [SD], 33.8 [10.7] years), all of whom tested negative for IgM and IgG antibodies to
SARS-CoV-2. Individuals were excluded if they had current infectious, inflammatory, or
immune-related diseases.
Preparation and administration of QFPDT
Twenty-one kinds of traditional Chinese herbs for the QFPDT formula were purchased from
Shanghai Ruisha Comlat Co., Ltd. (Shanghai, China). The herbs were mixed in accordance
with the above-mentioned Chinese official guideline; however, we reduced the dose of each
herb to 1/30 to explore the feasibility of prophylactic use and avoid possible adverse effects
such as palpitation. The QFPDT decoction was prepared by boiling the mixed herbs in 600
ml of water for 1 h before the first administration (day 1), divided into six aliquots and
stored at 4°C during the trial. The subjects were instructed to take an aliquot of the decoction
orally 40 min after breakfast and dinner for 3 days (day 1–3). Peripheral blood samples were
obtained 12 h before the first administration (day 0) and after the last administration (day 4).
Hematological, biochemical and cytokine analyses
Hematological and biochemical tests were outsourced to SRL, Inc. (Tokyo, Japan). Plasma
cytokines were quantified using V-PLEX Proinflammatory Panel 1 Human Kit (Meso Scale
Diagnostics) and Human IL-18 ELISA Kit (Abcam). All collected data (n = 18) were
subjected to the statistical analysis using a two-tailed paired t-test.
Results
Blood urea nitrogen, mean corpuscular volume, and mean corpuscular hemoglobin
concentration changed marginally within the normal ranges (Table 1), but there were no
significant differences in other hematological and biochemical measures between day 0 and
medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20146175.this version posted July 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.
5
day 4. Notably, we found that the plasma levels of proinflammatory cytokines IL-1β, IL-18,
TNF-α, and IL-8 in day 4 were significantly higher than those in day 0 (IL-1β: 0.173 vs.
0.558, P = 0.0198; IL-18: 119 vs. 269, P = 0.0207; TNF-α: 1.82 vs. 2.36, P = 0.00536; IL-8:
291 vs. 663, P = 0.00184; Table 1). These cytokines were increased in 14 (77.8%), 15
(83.3%), 15 (83.3%), and 17 (94.4%) out of 18 subjects, respectively (Fig. 1). We also found
a slight increase in IL-6 (0.552 vs. 0.852), which is known to be closely associated with the
severity and mortality of COVID-19 [4–6]; however, the difference was not statistically
significant (P = 0.147). No apparent adverse effects were observed during the trial.
Discussion
Inflammation is a host defense mechanism against invading pathogens. Acute inflammatory
responses to the infection of single-stranded RNA (ssRNA) viruses such as SARS-CoV-2 are
triggered by foreign ssRNA sensors such as Toll-like receptor (TLR) 7, TLR8 and NOD-like
receptor, pyrin domain containing 3 (NLRP3) [7,8]. TLR7 and TLR8 are expressed mainly
in dendritic cells and monocytes/macrophages and induce the expression of proinflammatory
cytokines, including TNF-α and IL-6, in response to viral ssRNA [7]. NLRP3, when it
recognizes viral ssRNA in the cytoplasm, initiates the inflammasome-mediated processing of
pro-IL-1β and pro-IL-18 to release active IL-1β and IL-18 [7,9]. We speculate that QFPDT
may create a blood cytokine conditions similar to those in acute antiviral immune responses.
TNF-α and IL-1β are well-recognized central regulators of acute inflammatory
reactions. In particular, IL-1β activates neutrophil functions, such as migration, superoxide
production, and survival. Conversely, neutrophils stimulates alveolar macrophages to release
IL-1β during respiratory viral infection [10]. IL-18 also has diverse bioactivities; it activates
both Th1–mediated cellular and Th2–mediated humoral immunity and enhances the
cytotoxic activity of NK cells, which attack and eliminate virus-infected cells [9]. IL-8 is
medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20146175.this version posted July 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.
6
produced by a various types of cells such as monocytes/macrophages, fibroblasts and
endothelial cells and serves as a potent chemotactic factor that induces the migration of
neutrophils to the site of infection and enhances its phagocytic activity. In the respiratory
system, IL-8 is produced by alveolar and airway epithelial cells, and IL-8–dependent
inflammation in the lung is the first line of innate immune responses against respiratory
pathogens [11].
In our trial, QFPDT had no significant effects on the plasma levels of IL-6 and IFN-γ.
An excessive increase in IL-6 leads to severe immune dysregulation in COVID-19 patients
[4–6]. IFN-γ is a critical mediator of Th1–dependent cellular immunity; however, a recent
study has shown that IFN-γ stimulates the expression of the SARS-CoV-2 receptor ACE2
and thereby facilitates the viral entry into host airway epithelial cells [12]. The
ineffectiveness of QFPDT on IL-6 and IFN-γ may thus be a preferable property for the
prevention of CIVID-19.
In conclusion, our findings suggest that even a lower dose of QFPDT can upregulate a
subset of proinflammatory cytokines without apparent adverse effects. However, since
COVID-19 patients present with a broad range of inflammatory symptoms [4–6], this
immunopharmacological property of QFPDT is apparently contradictory to its therapeutic
benefits in the previous clinical trials [1–3]. We should therefore be careful in its
prophylactic use in uninfected individuals until we have a better understanding of the
pharmacological action of QFPDT through further clinical studies with larger cohorts.
Funding: This research did not receive any specific grant from funding agencies in the
public, commercial, or not-for-profit sectors.
medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20146175.this version posted July 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.
7
Disclosure of Potential Conflict of Interest: Y.K., K.A., K.K., M.M., and T.E. are
employees of Takanawa Clinic. T.A. and T.N. have advisory roles in conducting clinical
research in Takanawa Clinic and receive advisory fees from Takanawa Clinic.
Data Availability Statement: The data that support the findings of this study are available
on request from the corresponding author, T.N. The data are not publicly available due to
their containing information that could compromise the privacy of research participants.
Author Contributions: Y.K., K.A., K.K., M.M., and T.E. contributed to the conception and
design of the study, contributed to data acquisition, analysis, and interpretation, and critically
revised the manuscript for important intellectual content. T.A. contributed to the conception
and design of the study and critically revised the manuscript for important intellectual
content. T.N. contributed to the conception and design of the study, contributed to data
analysis and interpretation, and drafted the manuscript. All authors gave final approval of the
submitted version of the manuscript and agree to be accountable for all aspects of the work.
References
[1] Ren JL, Zhang AH, Wang XJ. Traditional Chinese medicine for COVID-19
treatment. Pharmacol Res. 2020;155:104743.
[2] Yang Y, Islam MS, Wang J, Li Y, Chen X. Traditional Chinese Medicine in the
Treatment of Patients Infected with 2019-New Coronavirus (SARS-CoV-2): A
Review and Perspective. Int J Biol Sci. 2020;16(10):1708-1717.
[3] Cao P, Wu S, Wu T, et al. The important role of polysaccharides from a traditional
Chinese medicine-Lung Cleansing and Detoxifying Decoction against the COVID-
19 pandemic. Carbohydr Polym. 2020;240:116346.
[4] Giamarellos-Bourboulis EJ, Netea MG, Rovina N, et al. Complex Immune
Dysregulation in COVID-19 Patients with Severe Respiratory Failure. Cell Host
medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20146175.this version posted July 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.
8
Microbe. 2020;27(6):992-1000.e3.
[5] Aziz M, Fatima R, Assaly R. Elevated interleukin-6 and severe COVID-19: A
metaanalysis. J Med Virol. 2020;10.1002/jmv.25948.
[6] Blanco-Melo D, Nilsson-Payant BE, Liu WC, et al. Imbalanced Host Response to
SARS-CoV-2 Drives Development of COVID-19. Cell. 2020;181(5):1036-1045.e9.
[7] Chen N, Xia P, Li S, Zhang T, Wang TT, Zhu J. RNA sensors of the innate immune
system and their detection of pathogens. IUBMB Life. 2017;69(5):297-304.
[8] Moreno-Eutimio MA, López-Macías C, Pastelin-Palacios R. Bioinformatic analysis
and identification of single-stranded RNA sequences recognized by TLR7/8 in the
SARS-CoV-2, SARS-CoV, and MERS-CoV genomes. Microbes Infect.
2020;22(45):226-229.
[9] Slaats J, Ten Oever J, van de Veerdonk FL, Netea MG. IL-1β/IL-6/CRP and IL-
18/ferritin: Distinct Inflammatory Programs in Infections. PLoS Pathog.
2016;12(12):e1005973.
[10] Peiró T, Patel DF, Akthar S, et al. Neutrophils drive alveolar macrophage IL-1β
release during respiratory viral infection. Thorax. 2018;73(6):546-556.
[11] Reynolds CJ, Quigley K, Cheng X, et al. Lung Defense through IL-8 Carries a Cost
of Chronic Lung Remodeling and Impaired Function. Am J Respir Cell Mol Biol.
2018;59(5):557-571.
[12] Ziegler CGK, Allon SJ, Nyquist SK, et al. SARS-CoV-2 Receptor ACE2 Is an
Interferon-Stimulated Gene in Human Airway Epithelial Cells and Is Detected in
Specific Cell Subsets across Tissues. Cell. 2020;181(5):1016-1035.e19.
medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20146175.this version posted July 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.
9
Figure Legend
Figure 1. Changes in the plasma IL-1β, IL-18, TNF-α, and IL-8 levels before and after lowdose administration of QFPDT.
medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20146175.this version posted July 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.
10
Table 1. Hematological and cytokine changes in QFPDT-administered healthy individuals.
Complete blood count Red blood cell count (x 104/μl) 452 44.4 449 45.3 -3.06 -11.7–5.57 0.465 Hemoglobin (g/dl) 13.5 1.78 13.5 1.79 -0.0556 -0.347–0.236 0.692 Hematocrit (%) 40.8 4.58 41.4 4.68 0.578 -0.370–1.53 0.216 MCV (fl) 90.4 6.31 92.3 6.69 1.93 0.908–2.96 0.000971*** MCH (pg) 29.9 2.60 30.0 2.72 0.106 -0.0878–0.299 0.265 MCHC (%) 33.1 1.39 32.5 1.38 -0.600 -0.946 to -0.254 0.00194** White blood cell count (/μl) 6160 1240 5890 1270 -267 -692–158 0.203 Platelet count (x 104/μl) 25.1 3.50 24.5 3.74 -0.511 -1.40–0.373 0.239 White blood cell differential Neutrophils (%) 59.2 6.86 57.9 7.83 -1.29 -4.04–1.46 0.337 Eosinophils (%) 1.77 1.28 1.81 1.20 0.0333 -0.374–0.441 0.865 Basophils (%) 0.461 0.215 0.511 0.249 0.0500 -0.0422–0.142 0.269 Monocytes (%) 5.00 1.10 5.09 1.10 0.0944 -0.381–0.569 0.680 Lymphocytes (%) 33.6 6.67 34.7 7.12 1.11 -1.30–3.53 0.345 Blood biochemistry AST (U/l) 17.9 4.02 18.8 4.22 0.833 -0.105–1.77 0.0782 ALT (U/l) 18.7 12.0 19.1 12.3 0.389 -0.866–1.64 0.522 γ-GT (U/l) 35.9 49.2 34.4 45.8 -1.50 -3.93–0.932 0.210 LDH (U/l) 155 21.0 159 19.3 4.50 -1.06–10.1 0.106 Albumin (g/dl) 4.76 0.248 4.71 0.271 -0.0500 -0.151–0.0513 0.312 Urea nitrogen (mg/dl) 13.4 3.50 12.2 2.46 -1.18 -2.13 to -0.235 0.0174* HDL cholesterol (mg/dl) 68.5 11.8 68.3 9.97 -0.222 -2.69–2.25 0.852 LDL cholesterol (mg/dl) 114 40.2 112 36.3 -1.72 -6.12–2.68 0.420 Triglycerides (mg/dl) 92.2 61.2 95.6 74.5 3.39 -35.8–42.6 0.857 CRP (mg/dl) 0.0489 0.0466 0.0606 0.0567 0.0117 -0.00846–0.0318 0.238 Cytokines IFN-γ (pg/ml) 3.51 2.87 4.51 2.94 1.00 -0.337–2.34 0.133 IL-6 (pg/ml) 0.552 0.531 0.852 1.01 0.301 -0.117–0.718 0.147 TNF-α (pg/ml) 1.82 0.851 2.36 1.33 0.539 0.183–0.896 0.00536** IL-1β (pg/ml) 0.173 0.574 0.558 0.924 0.386 0.0693–0.702 0.0198* IL-18 (pg/ml) 119 64.6 269 280 150 25.9–274 0.0207* IL-2 (pg/ml) 0.641 1.01 0.747 1.12 0.106 -0.0217–0.234 0.0981 IL-8 (pg/ml) 291 685 663 933 372 159–585 0.00184** IL-10 (pg/ml) 0.255 0.203 0.298 0.199 0.0421 -0.0635–0.148 0.412
MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin; MCHC: mean corpuscular hemoglobin concentration;
Parameters Day 0 Day 4 Mean ( Day 4)
– Mean ( Day 0) 95 % CI P - value Mean SD Mean SD
medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20146175.this version posted July 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.
11
AST: aspartate aminotransferase; ALT: alanine aminotransferase; γ-GT: γ-glutamyltransferase; LDH: lactate dehydrogenase; HDL: high-density lipoprotein; LDL: low-density lipoprotein; CRP: C-reactive protein; IFN: interferon; IL: interleukin; TNF-α: tumor necrosis factor-α; SD: standard deviation; CI: confidence interval. *P < 0.05, **P < 0.01, ***P < 0.001.
medRxiv preprint doi: https://doi.org/10.1101/2020.07.13.20146175.this version posted July 20, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
All rights reserved. No reuse allowed without permission.